Lessons (not) learned from mistakes about translation

Kozak, Marilyn

doi:10.1016/j.gene.2007.08.017

Cited by 21 publications

(21 citation statements)

References 112 publications

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“…However, this appears not to be the case, because the method works equally well when two or three different stop codons are inserted between the ORFs, and/or the ORFs are placed out-of-frame, and Western blotting detected no such fusion products. Thus, it seems most likely that APHVIII is translated by post-termination reinitiation (Kozak 2007; Skabkin et al 2013), in which the ribosome remains associated with the mRNA after termination, continues scanning, and reinitiates translation at a downstream (or, occasionally, upstream: Skabkin et al 2013) AUG. Such events have been well documented for transcripts that have naturally occurring upstream ORFs (uORFs) in many organisms (Wang and Wessler 1998; Kozak 2007; Hinnebusch and Lorsch 2012), and many annotated transcripts in the Chlamydomonas genome also have uORFs, although it is not yet known how many of these are actually translated (Cross 2016).…”

Section: Discussionmentioning

confidence: 99%

“…Most uORFs in these cases are very short ( e.g. , 3–4 codons for yeast GCN4 and 38 codons for maize R ), and a negative correlation between uORF length and reinitiation efficiency at the downstream AUG has been reported (Kozak 2007). Thus, it is somewhat surprising that the long “uORFs” in the one-promoter constructs used in this study ( e.g.…”

Section: Discussionmentioning

confidence: 99%

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Robust Transgene Expression from Bicistronic mRNA in the Green AlgaChlamydomonas reinhardtii

Onishi

Pringle

2016

G3 Genes|Genomes|Genetics

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The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs) has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Robust Transgene Expression from Bicistronic mRNA in the Green AlgaChlamydomonas reinhardtii

Onishi

Pringle

2016

G3 Genes|Genomes|Genetics

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“…To identify an IRES function in viral and cellular mRNAs, mono-and dicistronic RNA-expressing plasmids have been extensively used during transfection studies. This approach, however, has been a subject of criticism due to expression via cryptic promoters and faulty transcription and splicing of the reporter constructs (51). To avoid spurious results generated by this method, we have used only in vitro transcribed capped and uncapped reporter RNAs for cell-free translation assays, transfection studies, and sucrose density gradient analyses.…”

Section: Discussionmentioning

confidence: 99%

Initiation Factor eIF2-independent Mode of c-Src mRNA Translation Occurs via an Internal Ribosome Entry Site

Allam¹,

Ali

2010

Journal of Biological Chemistry

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“…For example, it needs to be determined rigorously whether IRES elements are present in mitotically translated mRNAs as well as whether and to what extent IRES activity contributes to translation during mitosis. Several recently published reviews raise valid concerns about weak criteria used to claim that an mRNA carries an IRES and about insufficient reliability of the assays used to identify IRES activity in cellular mRNAs [69,70,134]. Moreover, it is important to exclude the possibility that alternative protein isoforms arise from alternative splicing, as opposed to IRES-mediated translational initiation [69].…”

Section: Concluding Remarks and Future Perspectivesmentioning

confidence: 99%

Translational regulation of the cell cycle: when, where, how and why?

Kronja

Orr‐Weaver

2011

Phil. Trans. R. Soc. B

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Translational regulation contributes to the control of archetypal and specialized cell cycles, such as the meiotic and early embryonic cycles. Late meiosis and early embryogenesis unfold in the absence of transcription, so they particularly rely on translational repression and activation of stored maternal mRNAs. Here, we present examples of cell cycle regulators that are translationally controlled during different cell cycle and developmental transitions in model organisms ranging from yeast to mouse. Our focus also is on the RNA-binding proteins that affect cell cycle progression by recognizing special features in untranslated regions of mRNAs. Recent research highlights the significance of the cytoplasmic polyadenylation element-binding protein (CPEB). CPEB determines polyadenylation status, and consequently translational efficiency, of its target mRNAs in both transcriptionally active somatic cells as well as in transcriptionally silent mature Xenopus oocytes and early embryos. We discuss the role of CPEB in mediating the translational timing and in some cases spindle-localized translation of critical regulators of Xenopus oogenesis and early embryogenesis. We conclude by outlining potential directions and approaches that may provide further insights into the translational control of the cell cycle.

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Lessons (not) learned from mistakes about translation

Cited by 21 publications

References 112 publications

Robust Transgene Expression from Bicistronic mRNA in the Green AlgaChlamydomonas reinhardtii

Robust Transgene Expression from Bicistronic mRNA in the Green AlgaChlamydomonas reinhardtii

Initiation Factor eIF2-independent Mode of c-Src mRNA Translation Occurs via an Internal Ribosome Entry Site

Translational regulation of the cell cycle: when, where, how and why?

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